GSM6620662: HEY1-GFP, replicate 1, CUT&RUN; Mus musculus; OTHER
CUT&RUN was performed as described (Meers et al., 2019; Peter J. Skene and Henikoff, 2017), with protocol and buffers detailed in Protocol 2: High Ca2+/low salt (https://www.protocols.io/view/cut-amp-run-targeted-in-situ-genome-wide-profiling-14egnr4ql5dy/v3). Briefly, MYCL-GFP, HEY1-GFP or NLS-GFP transduced mTECs were dissociated in 0.05% trypsin-EDTA in cell dissociation buffer for 15 min at 37°C. Cells were collected in in mTEC Basic medium with 2% Nu-Serum, washed twice in 5 mL of CUT&RUN Wash Buffer, and resuspended in CUT&RUN Wash Buffer. One million cells of each sample were aliquoted in 1.5 mL Eppendorf tubes and brought up to a volume of 1 mL with CUT&RUN Wash Buffer. Cells were incubated with 10 uL activated concavalin A beads (Bangs Laboratories, Cat #BP531) at room temperature for 10 min with gentle rocking. Cells were incubated overnight with anti-GFP (Abcam, Cat #ab290) diluted 1:100 in 0.05% digitonin in CUT&RUN Wash Buffer.  Following two washes in CUT&RUN Wash Buffer, samples were incubated for 1 hour at room temperature in 1:100 rabbit anti-guinea pig IgG in 0.05% digitonin in CUT&RUN Wash Buffer. Samples were incubated with Protein A/G-MNase fusion protein (Addgene, Cat #123461) at 700 ng/mL for 1 hour at 4°C. Following two washes in 0.05% digitonin in CUT&RUN Wash Buffer and one wash in CUT&RUN Low-Salt Rinse Buffer, samples were digested for 10 min at 0°C in cold CUT&RUN Incubation Buffer. Chromatin fragments were released by incubation at 37°C for 30 min and then collected by phenol-chloroform extraction. Library preparation was performed with the NEB Next Ultra II DNA Library Prep Kit for Illumina (NEB, Cat #E7645S) using 25 uL of CUT&RUN DNA as input, as detailed by Liu N. (https://www.protocols.io/view/library-prep-for-cut-amp-run-with-nebnext-ultra-ii-kxygxm7pkl8j/v2)